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Central Dogma DNA is the genetic material. It is used to make RNA, the “transport form” of genetic information, which travels to the ribosome. “Reading” the information in RNA, the ribosome synthesizes protein, which goes on to form or do the work of the cell.DNA-RNA-Protein

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Who figured it all out? 1927: Griffith described transmission of virulence from dead virulent bacteria to live avirulent bacteria 1944: Avery, McCleod and McCarty demonstrate DNA is the transforming principle in bacteria. 1952: Hershey and Chase tracked radiolabeled DNA and protein as viruses infected proteins.

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Hershey and Chase 32 P to label viral DNA 35 S to label viral protein Let the virus infect the bacteria and see where the radioactivity goes.

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It’s a quiz… what was their hypothesis? If DNA is the genetic material, then the 32 P will move into the bacterial cell. Alternatively, if protein is the genetic material, then the 35 S will move into the bacterial cell.

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So what happened? Most of the 32 P-DNA transferred into the bacteria following viral adsorption, while most of the 35 S- protein stayed outside the bacteria and was recovered in the empty phage coats stripped off the infected bacteria. The viruses that were produced inside the bacteria contained 32 P but not 35 S.

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The Exception: RNA Viruses Some viruses (including the AIDS virus) use RNA as their genetic material. When these viruses infect a host cell, they typically make a DNA copy of their genome that then is inserted into the host genome (latent cycle) or is used to direct the lytic cycle. reverse transcriptase The viral enzyme is called reverse transcriptase because it makes a DNA copy from an RNA template.

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Features of DNA The DNA structure is such that the bases (adenine, guanine, cytosine and thymine) of opposing strands face each other. Hydrogen bonds form between the bases, holding the strands together.

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Hydrogen Bonds….remember your chemistry?? Hydrogen bond: a weak association between a covalently bonded hydrogen atom and an unshared electron pair from another covalently bonded atom (in this case oxygen and nitrogen) Alone, they’re pretty wimpy, but thousands in a row create a very stable force holding the two strands of DNA together.

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Replication “It has not escaped our notice that the specific pairing we have postulated immediately suggests a possible copying mechanism for the genetic material.” J.D. Watson and F.H.C. Crick, 1953

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How might DNA Replicate? The complementary nature of DNA lends clues to how DNA might copy itself: 1. Conservative: “old” double strand goes to one daughter strand intact, other daughter cell gets a new copy. 2. Dispersive: Parental strands are dispersed into two new double helices following replication. Both daughter cells would receive “old” and “new,” but would involve cleavage of the parental strands. Most complicated and least likely

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How might DNA Replicate? 3. Semi-conservative: Each daughter cell receives one new strand, one old strand. Each strand serves as a template to synthesize the complementary strand.

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Meselson-Stahl Experiment (1958) Strongly supported semiconservative hypothesis…. Grew E. coli in medium that contained only 15 NH 4 Cl as the nitrogen source for many generations such that all the N within the bacteria was radioactive.

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Meselson-Stahl Experiment (1958) The natural form of N is 14 N, which is lighter than 15 N. DNA can be separated by weight using centrifugation DNA made with 15 N is heavier than DNA made with 14 N and will form a discrete band from 14 N-DNA

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Meselson-Stahl Experiment (1958) When the E.coli were switched to non-radioactive N- source ( 14 N), all “new” DNA would be made with 14 N. After one generation, there was only one band of intermediate density (1:1 15 N : 14 N) (If replication were conservative, there would be two bands)

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Meselson-Stahl Experiment (1958) - 15 N-DNA/ 15 N-DNA band was not observed again (purely radioactive molecule was not preserved) Replication in eukaryotes was later proved to occur by the same means.

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Models for Recombination Based on proposals put forth by Robin Holliday and Harold Whitehouse in 1964. Depend on complementarity of DNA strands Rely on enzymatic processes

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Basic Model Two pair DNA duplexes An endonuclease nicks one strand of each (breaks the phosphodiester bond in the backbone) The ends of the strands are displaced The homologous regions of the displaced strands pair up Ligase seals the nicks

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Basic Model heteroduplex The hybrid duplex formed is a heteroduplex :

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Basic Model branch migration The cross bridge then migrates down the strand in a process called branch migration as hydrogen bonds are broken, then reformed.

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Basic Model Holliday Structure If the duplexes separate and the structure rotates 180º, an intermediate structure is formed called a Holliday Structure.

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Evidence for these models 1. Visualization of the intermediate planar Holliday structure. 2. Discovery of Rec A protein in E. coli that promotes exchange of reciprocal single-stranded DNA molecules. 3. Discovery of other enzymes essential to nicking, unwinding and ligation of DNA.

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